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Testing the effect of pollen exine rupture on metabarcoding with Illumina sequencing
Stephanie J Swenson1, Birgit Gemeinholzer1
1Systematic Botany, Justus Liebig University Gießen, Giessen, Germany.
Plos One
|February 2, 2021
Summary
Optimizing pollen metabarcoding requires careful DNA extraction. Moderate exine rupture (33-67%) and shorter lysis times improve DNA yield and sequencing accuracy for reliable taxonomic identification.
Area of Science:
- Environmental science
- Molecular biology
- Bioinformatics
Background:
- Pollen metabarcoding offers high taxonomic resolution for ecological and public health studies.
- Varied methodologies in pollen metabarcoding hinder result comparability and reliability.
Purpose of the Study:
- To investigate the impact of exine rupture levels and lysis incubation time on DNA extraction and sequencing performance.
- To optimize DNA extraction protocols for pollen metabarcoding.
Main Methods:
- Examined 15 plant species with diverse pollen characteristics to assess exine rupture effort.
- Created mock communities using DNA from 4 exine rupture levels (0-100%) and 2 lysis times (2h, 24h).
- Performed Illumina sequencing on three DNA barcode regions (rbcL, ITS1, ITS2) with varying primer pairs.
Main Results:
- Increased exine rupture correlated positively with DNA yield.
- Extended lysis incubation time decreased DNA yield.
- Moderate exine rupture (33-67%) yielded better sequencing performance than 0% or 100% rupture.
- Increased lysis incubation time negatively impacted results.
Conclusions:
- Optimal pollen metabarcoding performance is achieved with moderate exine rupture.
- Shorter lysis incubation times are recommended for improved DNA extraction and sequencing accuracy.
- Standardized DNA extraction protocols are crucial for reliable pollen metabarcoding results.
